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Identification of Two Distinct Mechanisms of Phagocytosis Controlled by Different Rho GTPases

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Science  27 Nov 1998:
Vol. 282, Issue 5394, pp. 1717-1721
DOI: 10.1126/science.282.5394.1717

Abstract

The complement and immunoglobulin receptors are the major phagocytic receptors involved during infection. However, only immunoglobulin-dependent uptake results in a respiratory burst and an inflammatory response in macrophages. Rho guanosine triphosphatases (molecular switches that control the organization of the actin cytoskeleton) were found to be essential for both types of phagocytosis. Two distinct mechanisms of phagocytosis were identified: Type I, used by the immunoglobulin receptor, is mediated by Cdc42 and Rac, and type II, used by the complement receptor, is mediated by Rho. These results suggest a molecular basis for the different biological consequences that are associated with phagocytosis.

Phagocytosis is the process by which cells recognize and engulf large particles (>0.5 μm) and is important to host defense mechanisms as well as to tissue repair and morphogenetic remodeling. Two of the best characterized phagocytic receptors in macrophages, the complement receptor 3 (CR3) and Fc gamma receptors (FcγRs), are involved in the uptake of opsonized microorganisms during infection. CR3 binds C3bi on complement-opsonized targets, whereas FcγRs bind to immunoglobulin G (IgG)–coated targets. Phagocytosis by both types of receptors is driven by the reorganization of filamentous actin (F-actin), but the mechanisms of uptake appear to be different (1, 2). First, FcγR-mediated uptake is accompanied by pseudopod extension and membrane ruffling, whereas complement-opsonized targets sink into the cell, producing little protrusive activity (3). Second, FcγR ligation is accompanied by the activation of the respiratory burst (to produce reactive oxygen species) and by the production of arachidonic acid metabolites and cytokines, such as tumor necrosis factor–α. C3bi-dependent uptake occurs in the absence of any of these proinflammatory signals (4–6).

The Rho family of small guanosine triphosphatases (GTPases) is involved in the reorganization of filamentous actin structures in response to extracellular stimuli (7). Rho induces the assembly of contractile actomyosin filaments, whereas Rac and Cdc42 control actin polymerization into lamellipodial and filopodial membrane protrusions, respectively (8, 9). In addition, these GTPases can affect gene transcription [through the activation of nuclear factor kappa B, through the c-Jun NH2-terminal kinase (JNK), and through the p38 mitogen-activated protein kinase (MAPK)], and Rac regulates the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex that is responsible for the respiratory burst (10, 11). We have, therefore, analyzed the relative roles of Rho, Rac, and Cdc42 in FcγR- and CR3-mediated phagocytosis.

Quiescent, serum-starved Swiss 3T3 fibroblasts provide a simple model for studying the formation of polymerized actin structures that are induced by extracellular stimuli (8, 9). To analyze the effects of FcγR and CR3 receptor activation on actin, we microinjected quiescent cells with either a plasmid encoding the single FcγRIIA chain or with a combination of plasmids encoding the two chains of the CR3 integrin receptor (CD11b and CD18) (12). Surface expression of the receptors, visualized by immunofluorescence 2 hours after microinjection, did not in itself affect cell morphology or F-actin distribution (Fig. 1A). However, antibody cross-linking of FcγRII resulted in the formation (within 10 min) of filopodia, accompanied by localized ruffles (Fig. 1B). After 30 min, cells harboring cross-linked FcγR showed a contracted morphology and contained stress fibers. Antibody cross-linking of CR3 induced rapid cell contraction and F-actin reorganization into stress fibers with no evidence of filopodia or lamellipodial protrusions (Fig. 1C).

Figure 1

Activation of Rho GTPases by FcγR and CR3 receptors in Swiss 3T3 fibroblasts and in COS cells. (A through D) Cross-linking of FcγR or CR3 receptors activates Cdc42 or Rho in Swiss 3T3 fibroblasts. F-actin distribution in microinjected (12) subconfluent serum-starved fibroblasts expressing FcγRII [(A), (B), and (D)] or CR3 (C), which were subjected to receptor crosslinking for 10 min [(B) and (D)] or 30 min (C), is shown. In (D), cells were coinjected with N17Cdc42. Scale bars, 10 μm. (E) Rho GTPases control FcγR- and CR3-mediated phagocytosis in COS cells. COS cells were cotransfected (13) with phagocytic receptors [that is, FcγRIIA or CR3 (CD11b+CD18)] and with either an empty vector (−) or with vectors expressing Myc-tagged versions of C3 transferase (C3), dominant negative Rac (N17Rac), or dominant negative Cdc42 (N17Cdc42). Phagocytosis assays were then performed (n ≥ 3) with appropriately opsonized RBCs; ≥100 transfected cells were scored for their ability to bind (right) or phagocytose (left) RBCs (mean ± SEM).

To determine whether changes in the actin cytoskeleton were mediated by the activation of Rho GTPases, we expressed the phagocytic receptors along with inhibitors of Rho, Rac, or Cdc42. Coinjection of a dominant negative Cdc42 construct blocked the formation of all filamentous actin structures upon FcγRIIA ligation (Fig. 1D), whereas dominant negative Rac blocked ruffling and stress fibers but not filopodia. With CR3, dominant negative Cdc42 or Rac constructs had no effect on the induced actin changes, whereas the Rho inhibitor (C3 transferase) blocked all actin changes. Thus, FcγRIIA cross-linking specifically results in the activation of Cdc42, which in turn activates a previously described Cdc42-Rac-Rho cascade (8,9). However, cross-linking of the integrin receptor CR3 activates only Rho.

As a first step toward analyzing the role of Rho GTPases in phagocytosis, plasmids encoding FcγRII or CR3 receptors were transfected into COS cells. Cells were then presented with red blood cells (RBCs) that were opsonized with either IgG (for FcγR) or C3bi (for CR3), and the efficiency of particle internalization was determined (13, 14). Ninety-seven percent of FcγR-expressing COS cells were able to bind IgG-opsonized cells, and ∼60% of expressing cells contained one or more internalized particles (Fig. 1E). Cotransfection of the two chains of the CR3 receptor resulted in 70% of CR3-expressing cells binding to opsonized particles, and ∼50% of these showed one or more internalized particles. Both chains of the CR3 receptor were needed for the efficient binding and ingestion of complement-opsonized cells (Fig. 1E). Next, both phagocytic receptors were cotransfected with dominant negative versions of Rho, Rac, or Cdc42 or with the specific Rho inhibitor (C3 transferase). None of the inhibitors had any substantial effect on the binding of opsonized RBCs to either CR3- or FcγR-expressing COS cells (Fig. 1E). Dominant negative Cdc42 and dominant negative Rac (Fig. 1E) showed no inhibitory effect on the phagocytic behavior of CR3-transfected COS cells, whereas both dominant negative Rho and C3 transferase showed essentially complete inhibition of CR3-mediated phagocytosis (Fig. 1E). For FcγR-mediated phagocytosis, dominant negative Rho and C3 transferase had no effect, whereas dominant negative Cdc42 and dominant negative Rac abolished particle internalization (Fig. 1E). Thus, CR3-mediated phagocytosis in COS cells is mediated by Rho, and FcγR-mediated phagocytosis is mediated by a combination of Cdc42 and Rac.

To determine whether Rho GTPases control phagocytosis in professional phagocytic cells, we treated the mouse macrophage cell line J774 with toxin B from Clostridium difficile, an inhibitor of all members of the Rho family (15). A pretreatment (2 hours) with toxin B inhibited (in a dose-dependent manner) both CR3- and FcγR-mediated phagocytosis but had no effect on the initial binding of opsonized targets to J774 macrophages (15). To identify the specific GTPases involved, we microinjected J774 cells with plasmids that encoded inhibitors of Rho, Rac, and Cdc42 (16). Representative examples of the results that were obtained for FcγR-mediated phagocytosis are shown (Fig. 2, A through H). C3 transferase–expressing cells (Fig. 2, C, D, and I) or dominant negative Rho–expressing cells were as competent as control cells (Fig. 2, A, B, and I) at internalizing IgG-opsonized RBCs, which appear as swollen particles in vacuoles (14). In contrast, macrophages expressing dominant negative Rac (Fig. 2, E, F, and I) or dominant negative Cdc42 (Fig. 2G, H and I) were unable to carry out FcγR-mediated phagocytosis, as attested by the crenated morphology of the RBCs (14). As an alternative to using dominant negative proteins, the Cdc42-binding domain of a specific Cdc42 target protein, Wiscott-Aldrich syndrome protein (WASP) (17), was also used, which completely prevented FcγR-mediated phagocytosis (Fig. 2I). In agreement with the results presented here, an earlier report showed that both Rac and Cdc42 are required for FcγR uptake (18). Another report, however, suggested that Rho is also required for FcγR uptake, but in those experiments, Rho inhibition prevented even the binding of opsonized particles (19).

Figure 2

(left).Cdc42 and Rac are necessary for FcγR-mediated phagocytosis in macrophages. (A through H) FcγR-mediated phagocytosis in J774.A1 cells that were microinjected with biotin dextran and control plasmid [(A) and (B)], Myc-tagged C3 transferase [(C) and (D)], N17Rac [(E) and (F)], or N17Cdc42 [(G) and (H)]. Microinjected cells were detected by costaining with cascade blue–conjugated avidin (A) or 9E10 anti-Myc [(C) (E), and (G)], and RBCs were visualized with anti-rabbit IgG [(B), (D), (F), and (H)]. Scale bar, 10 μm. (I) Quantitation of the J774.A1 cell ability to bind (right) and phagocytose (left) IgG-opsonized RBCs and the effect of an empty vector (−) or the effect of microinjected expression vectors encoding C3 transferase (C3), dominant negative Rac (N17Rac), dominant negative Cdc42 (N17Cdc42), or Cdc42-binding domain (amino acids 201 through 321) of WASP. Data shown are the mean ± SEM of three to five independent experiments.

Representative examples of the results that were obtained for CR3-mediated phagocytosis are shown (Fig. 3). In this case, macrophages expressing dominant negative Rac (Fig. 3, E, F, and I) or dominant negative Cdc42 (Fig. 3, G through I) were as competent as control cells [Fig. 3, A, B, and I (left)] at internalizing complement-opsonized RBCs. C3 transferase–expressing cells (Fig. 3I) or dominant negative Rho–expressing cells (Fig. 3, C and D), however, were unable (up to 90% inhibition) to carry out CR3-mediated phagocytosis. Thus, FcγR receptors activate Cdc42 and Rac, and both these GTPases (but not Rho) are required for phagocytosis. CR3-mediated phagocytosis occurs through a distinct cellular mechanism and is dependent on Rho (but not dependent on Rac or Cdc42).

Figure 3

(right). Rho is necessary for CR3-mediated phagocytosis in macrophages. (A throughH) CR3-mediated phagocytosis in J774.A1 cells that were microinjected with biotin dextran and control plasmid [(A) and (B)], Myc-tagged N19Rho [(C) and (D)], N17Rac [(E) and (F)], or N17Cdc42 [(G) and (H)]. Microinjected cells were detected by costaining with cascade blue–conjugated avidin (A) or 9E10 [(C), (E), and (G)], and RBCs were visualized with anti-rabbit IgG [(B), (D), (F), and (H)]. Scale bar, 10 μm. (I) Quantitation of the microinjected J774.A1 cells ability to bind (right) and phagocytose (left) C3bi-opsonized RBCs and the effect of an empty vector (−) or the effect of microinjected constructs encoding C3 transferase (C3), dominant negative Rac (N17Rac), or dominant negative Cdc42 (N17Cdc42). Data shown are the mean ± SEM of three to five independent experiments.

To determine whether Rho GTPases are differentially recruited to phagosomes surrounding IgG- or complement-opsonized particles, we analyzed their association with internalized particles. In the absence of antibody reagents that were sufficiently sensitive to visualize all three endogenous Rho GTPases in J774 macrophages, the behavior of wild-type, tagged GTPases was analyzed in the COS cell phagocytosis assay. Opsonized particles that were internalized through either CR3 or FcγR expressed in COS cells were both associated with F-actin. Coexpression of the FcγR receptor with Myc-tagged versions of wild-type Rho (Fig. 4B), Rac (Fig. 4D), or Cdc42 revealed that all three GTPases were recruited along with actin (Fig. 4, A and C) to the IgG-opsonized particles. However, when the experiment was repeated with CR3-expressing COS cells, only Rho (Fig. 4F), and not Rac (Fig. 4H) or Cdc42, colocalized with F-actin (Fig. 4, E and G).

Figure 4

Differential recruitment of Rho and Rac to nascent phagosomes during complement- and IgG-dependent phagocytosis. COS cells were cotransfected with (A through D) FcγRIIA or (E throughH) CR3 and wild-type Myc-tagged versions of Rho [(A), (B), (E), and (F)] and Rac [(C), (D), (G), and (H)]. Cells were challenged with the appropriate opsonized targets, and immunofluorescence was performed to detect Myc, that is, Rho GTPases [(B), (D), (F), and (H)] and F-actin [(A), (C), (E), and (G)]. Cells that were cotransfected with wild-type Rac [(C), (D), (G), and (H)] were examined with confocal microscopy. Scale bar, 10 μm.

Thus, Cdc42 is activated after FcγRII ligation, and this results in the activation of a Rac-Rho cascade and the concomitant association of all three GTPases with the phagosome. In this case, both Cdc42 and Rac, but not Rho, activities are required for particle internalization. By contrast, CR3-induced phagocytosis only results in the activation of Rho, and only Rho is recruited to phagosomes surrounding the complement-opsonized particles. The fact that Rho, Rac, and Cdc42 each interact with distinct effector molecules and affect the assembly and organization of filamentous actin in very different ways suggests that the biochemical mechanisms of particle uptake are quite different (20). We propose that the Cdc42/Rac-dependent uptake (typified by FcγR) be termed type I phagocytosis and the Rho-dependent uptake (typified by CR3) be termed type II. The recruitment of Rho, Rac, and Cdc42 in type I (but only Rho in type II) phagocytosis suggests a molecular explanation not only for the well-known morphological differences observed between FcγR- and CR3-mediated phagocytosis but, more important, for the different associated biological responses (2–6). Because Rac is an essential regulatory component of the NADPH oxidase enzyme complex and because both Rac and Cdc42 activate the JNK and p38 MAPK pathways, the reason why type I, but not type II, mediated phagocytosis is accompanied by an inflammatory response could be explained (11, 21). Finally, type II phagocytosis provides a possible explanation for the lack of an inflammatory response associated with the uptake of apoptotic cells and with the invasion of macrophages by pathogenic microorganisms such as Leishmania major andMycobacterium leprae (22).

  • * To whom correspondence should be addressed. E-mail: alan.hall{at}ucl.ac.uk

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